Recent work has established that specialized cell surface receptors (termed here selectins) on endothelial cells and various circulating cells are involved in a number of intercellular interactions. For instance, an adhesion molecule on the surface of leukocytes, lymphocyte homing receptor (LHR), is known to be involved in the adhesive interactions of leukocytes with the endothelial lining of blood vessels. This adhesive interaction is a prerequisite for the movement of leukocytes from the blood to tissue sites where immune reactions and inflammatory reactions occur.
LHR (also known as gp90.sup.MEL, gp100.sup.MEL, gp110.sup.MEL, Mel-14 antigen, Leu8 antigen, TQ antigen, DREG antigen, LAM-1, selectin 1, LECAM-1, LEC-CAM-1, LEEK and L-Selectin depending on animal species, leukocyte, and laboratory preference) is expressed on the surface of leukocytes, such as, lymphocytes, neutrophils, monocytes, and eosinophils (Gallatin, et al., Nature 303:30 (1983) and Lewisohn, et al., J. Immunol. 138:4313 (1987), which are incorporated herein by reference). LHR is known to mediate the adhesion of lymphocytes to specialized endothelial cells in lymph nodes, leading to the migration of blood-borne lymphocytes into the lymph node. On neutrophils and monocytes, it mediates the early interaction of these cells with endothelium of blood vessels at sites of inflammation.
LHR is a lectin-like protein which performs its adhesive function recognizing carbohydrate-containing ligands on endothelial cells. Lectin-like receptors have also been found on endothelial cells and platelets. Endothelial leukocyte adhesion molecule-1 (ELAM-1) also known as CD62E and E-Selectin is present on endothelial cells and is involved in the recognition of various circulating cells by the endothelium. Granule membrane protein-140 (GMP-140) also known as CD62P and P-Selectin is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions.
Recent work has established that these receptors share certain structural features. Each of the receptors in this class is a glycoprotein with a lectin-like domain, a region with homology to epidermal growth factor, and a region with homology to complement regulatory proteins (see, Springer, Nature, 346:425, 1989, which is incorporated herein by reference). The term "selectin" is used herein to refer to this class of lectin-like receptors.
There is currently an interest in developing highly specific competitive inhibitors of selectin-mediated cellular adhesion. Such inhibitors are useful in therapeutic regimens to treat various selectin-mediated disease responses. The inhibitors could also be used to target other pharmaceutical compounds, such as anti-inflammatory agents or anti-oxidants, to the sites of injury.
In view of the above it is important to improve our understanding of the interaction of selectin receptors and their ligands. Thus, it is important to develop rapid, economical methods for identifying inhibitors of selectin-mediated interactions. In the past in vitro intercellular adhesion assays have been used to test inhibition (see, e.g., Stamper and Woodruff, J. Exp. Med. 144:828-833 (1976), which is incorporated herein by reference). These assays, however, are difficult to carry out and do not lend themselves to screening large numbers of test compounds. Comparisons of active compounds by quantitative dose-response studies is difficult using these assays.
Previous ELISA's for L-selectin have measured its interaction with PPME, sulfatide, or sLe.sup.x, Foxall, C., S. R. Watson, D. Dowbenko, C. Fennie, L. A. Lasky, M. Kiso, A. Hasegawa, D. Asa, and B. K. Brandley. (1992). The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis.sup.x oligosaccharide. J. Cell. Biol. 117:895-902. Imai, Y., L. A. Lasky, and S. D. Rosen. (1992). Further characterization of the interaction between L-selectin and its endothelial ligands. Glycobiology. 2:373-381. Imai, Y., D. D. True, M. S. Singer, and S. D. Rosen. (1990). Direct Demonstration off the lectin activity of gp90MEL, a lymphocyte homing receptor. J. Cell. Biol. 111:1225-1232. The first two carbohydrates are fortuitous ligands, and their interaction with L-selectin reflects only limited features of the carbohydrate specificity of the lectin. sLe.sup.x is part of the endogenous ligands for L-selectin (see above), but without sulfate has very low affinity for L-selectin. The use of sLe.sup.x in an ELISA is thus problematic.
The present invention is a highly sensitive assay for finding compounds which inhibit interaction between a selectin receptor and a natural ligand. The present assay may be easily and efficiently carried out without the need for radioactive materials.